Project Summary CD8+ T cells classically detect HIV-infected cells through presentation of viral peptides on MHC-I molecules. However, HIV mutates rapidly and can subvert CD8+ T cell recognition. Two recent studies suggest that non-classical mechanisms of HIV recognition exist, and that these mechanisms can curb viral replication. In the simian immunodeficiency (SIV) model of HIV infection, a CMV-vectored vaccine protected 50% of monkeys vaccinated and later challenged with SIV. This vaccine elicited unique CD8+ T cell responses of wide breadth and promiscuity that were MHC-II- and HLA-E- restricted. A subsequent study observed Class II- restricted CD8+ T cells in a small percent of chronic HIV elite controllers. When these cells were transformed into T cell clones, they killed autologous CD4+ T cells superinfected with HIV. The ability of non-canonically restricted CD8+ T cells to recognize infected targets via peptides presented on Class II indirectly suggests that HIV antigens are processed and presented on MHC II on CD4+ T cells. Moreover, the paucity of these non-canonically restricted CD8+T cells in normal immune responses suggests that these cells must exist at low frequencies in macaques and humans and are boosted under unique circumstances. This project thus seeks to characterize non-classical peptide presentation on HIV-infected CD4+ T cells, focusing on the MHC-II pathway, and how this presentation influences responding cell fate. The ultimate goal is to identify a novel mode of target cell recognition that bypasses classical HIV evasion paradigms and to assess whether Class II-mediated T-T cell interactions should be enhanced or inhibited for therapeutic purposes. The proposed study will address this goal via two specific aims: 1) Demonstrating that non-classical antigen processing occurs in CD4+ T cells (A) resulting in peptide presentation that can be recognized by other T cells (B); and 2) Analyzing the T cell response to HIV peptides presented by CD4+ T cells on MHC Class II. Aim 1A will use untargeted & targeted mass spectrometry to analyze the Class II HIV peptidome on infected CD4+ T cells, and Aim 1B will use tetramer-based enrichment and expansion to show T cell recognition of Class II-bound HIV peptides on CD4+ T cells. Aim 2 will use assays testing cytotoxic function and anergy induction to define the fate of responding T cells to Class II-bound HIV peptides presented on CD4+ T cells. This study has the potential to fundamentally change our understanding of the immune response to highly evasive pathogens. Novel effector/target cell interactions identified may illuminate the immune system's natural failure to control HIV and may enable improved vaccine design that can overcome the rapid evolution and evasive strategies of the virus. As incidence rates of HIV are not decreasing and 36 million individuals are currently infected, advances in preventative and therapeutic vaccines are sorely needed to curb this epidemic.